We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting to these compartments is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats. Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (GGAs), and the heteropentameric retromer complex. Proper sorting also requires the function of other components of the trafficking machinery that mediate vesicle tethering and fusion. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects (Hermansky-Pudlak syndrome) or pathogens' (HIV-1) exploitation of these proteins. [unreadable] [unreadable] The retromer complex is a sorting device that mediates retrograde transport from endosomes to the trans-Golgi network. This function is essential for many important physiological processes in higher eukaryotes, including lysosomal enzyme sorting, processing of the Alzheimer's disease amyloid precursor protein, and formation of morphogen gradients during development. The retromer comprises a membrane-binding subcomplex made up of two sorting nexin (SNX) subunits and a cargo-recognition subcomplex composed of Vps26, Vps29 and Vps35. We have collaborated with James Hurley (NIDDK) and Alasdair Steven (NIAMS) to solve the atomic structure of the Vps26-Vps29-Vps35 subcomplex. The structure revealed that Vps35 is a rod-shaped, alpha-helical solenoid onto which Vps26 and Vps29 assemble at either end of the rod. Vps26 resembles a clathrin adaptor known as arrestin, whereas Vps29 belongs to a family of metallophosphoesterases. This structure has multiple sites for binding SNX proteins and cargo, and can flex to conform to the curved shape of transport vesicles and tubules.[unreadable] [unreadable] We have recently found that the recruitment of the Vps26-Vps29-Vps35 subcomplex to membranes involves, in addition to the SNX subcomplex, the small GTP-binding protein Rab7. Perturbation of either the SNX subcomplex or Rab7 results in dissociation of the Vps26-Vps29-Vps35 subcomplex from membranes. Loss of retromer function leads to missorting of lysosomal enzymes and consequent accumulation of undegraded materials in lysosomes, a phenotype characteristic of lysosomal storage disorders.[unreadable] [unreadable] Endosomal transport carriers formed by the action of retromer must dock at and fuse with the trans-Golgi network in order to deliver their cargo. We have recently found that GARP, a multi-protein complex originally described in yeast, plays such a role in mammalian cells. Interference with GARP blocks the delivery of cargos such as mannose 6-phosphate receptors and Shiga toxin from endosomes to the trans-Golgi network, indicating that GARP has a general role in retrograde transport. A mutation in one of the GARP subunits, Vps54, has recently been identified in the Wobbler mouse mutant, an animal model of amyotrophic lateral sclerosis. We have found that the Wobbler mutation does not prevent the function of GARP in retrograde transport, suggesting that the disease is likely due to a more subtle defect in transport or to some other function of GARP.[unreadable] [unreadable] Rab GTPases and ubiquitination are additional factors that regulate transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by being both a guanine-nucleotide-exchange factor (GEF) for Rab5 and a substrate for ubiquitin (Ub) binding and conjugation. This past year we demonstrated that the ability of Rabex-5 to bind Ub is essential for its recruitment from the cytosol to endosomes, independently of its GEF activity and of Rab5. We also showed that monoubiquitinated Rabex-5 is enriched in the cytosol. These observations are consistent with a model whereby a cycle of Ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.